Live imaging of DORNRÖSCHEN and DORNRÖSCHEN-LIKE promoter activity reveals dynamic changes in cell identity at the microcallus surface of Arabidopsis embryonic suspensions

Key message Transgenic DRN::erGFP and DRNL::erGFP reporters access the window from explanting Arabidopsis embryos to callus formation and provide evidence for the acquisition of shoot meristem cell fates at the microcalli surface. Abstract The DORNRÖSCHEN (DRN) and DORNRÖSCHEN-LIKE (DRNL) genes encode AP2-type transcription factors, which are activated shortly after fertilisation in the zygotic Arabidopsis embryo. We have monitored established transgenic DRN::erGFP and DRNL::erGFP reporter lines using live imaging, for expression in embryonic suspension cultures and our data show that transgenic fluorophore markers are suitable to resolve dynamic changes of cellular identity at the surface of microcalli and enable fluorescence-activated cell sorting. Although DRN::erGFP and DRNL::erGFP are both activated in surface cells, their promoter activity marks different cell identities based on real-time PCR experiments and whole transcriptome microarray data. These transcriptome analyses provide no evidence for the maintenance of embryogenic identity under callus-inducing high-auxin tissue culture conditions but are compatible with the acquisition of shoot meristem cell fates at the surface of suspension calli

Transcriptomic Analysis Highlights Time-specific Embryonic Adaptation of Mice to the Lack of PrP

The physiological function of the PrP remains largely elusive. Its invalidation does not affect mouse survival and induces subtle phenotypes. To potentially assess this conundrum, we first comparatively analyzed the adult brain transcriptome of wild-type mice with that of transgenic mice invalidated at this locus either at the zygotic (Zürich PrP0/0 mice) or adult stages (NFH-Cre-Lox mice). Only subtle differences could be evidenced in the adult brains following microarray and QPCR analyses. When performed at an early adult stage, neuronal Prnp disruption appeared to sequentially induce an oxidative stress response and a nervous system remodeling, but it involved a limited number of only slightly modified genes. In sharp contrast, analysis at early embryonic stages, 7.5 and 8.5 dpc, just after the suspected normal time set of the Prnp locus activation, led to a transient perturbation of the transcriptome involving a larger number of genes and pointing to potential pathways related to the PrP physiological function. Overall, our data suggests an early adaptation of the mouse to the potentially detrimental lack of PrP during embryogenesis while its presence is less influential or redundant at later developmental stages

Profiling the onset of somatic embryogenesis in Arabidopsis

Background: Totipotency is the ability of a cell to regenerate a whole organism. Plant somatic embryogenesis (SE) is a remarkable example of totipotency because somatic cells reverse differentiation, respond to an appropriate stimulus and initiate embryo development. Although SE is an ideal system to investigate de-differentiation and differentiation, we still lack a deep molecular understanding of the phenomenon due to experimental restraints. Results: We applied the INTACT method to specifically isolate the nuclei of those cells undergoing SE among the majority of non-embryogenic cells that make up a callus. We compared the transcriptome of embryogenic cells to the one of proliferating callus cells. Our analyses revealed that embryogenic cells are transcriptionally rather than metabolically active. Embryogenic cells shut off biochemical pathways involved in carbohydrate and lipid metabolism and activate the transcriptional machinery. Furthermore, we show how early in SE, ground tissue and leaf primordia specification are switched on before the specification of a shoot apical meristem. Conclusions: This is the first attempt to specifically profile embryogenic cells among the different cell types that constitute plant in vitro tissue cultures. Our comparative analyses provide insights in the gene networks regulating SE and open new research avenues in the field of plant regeneration

Additional file 3: Table S2. of Profiling the onset of somatic embryogenesis in Arabidopsis

Differentially expressed genes between embryogenic cells and callus cells. (XLSX 942 kb

Additional file 4: Table S3. of Profiling the onset of somatic embryogenesis in Arabidopsis

Expression data concerning all the genes discussed in the main text. (XLSX 16 kb

Additional file 1: Figure S1. of Profiling the onset of somatic embryogenesis in Arabidopsis

ProLEC2:NTF line. (A) GFP fluorescence image of a ProLEC2:NTF zygotic embryo. Bar = 50 μm. The inset shows a cotyledon close-up (green channel: GFP, red channel: chlorophyll). (B-C) Transmitted light (B) and GFP fluorescence image (C) of an isolated ProLEC2:NTF zygotic embryo on 2,4-D. Bar = 100 μm. (D-E) Transmitted light (D) and GFP (E) fluorescence image of a ProLEC2:NTF callus on 2,4-D. Bar = 500 μm. (F-G) Transmitted light (F) and GFP (G) fluorescence image of a ProLEC2:NTF callus on 2,4-D free medium. Bar = 500 μm. Figure S2. Purification of nuclei from embryogenic callus using INTACT. (A) DAPI fluorescence image of beads and ProLEC2:NTF nuclei (in Pro35S:BirA background) isolated from embryogenic callus. Bar = 50 μm. (B-C) DAPI (B) and GFP (C) fluorescence image of a ProLEC2:NTF (in Pro35S:BirA background) nucleus surrounded by beads isolated from embryogenic callus. Bar = 10 μm. (D-E) DAPI fluorescence image of ProLEC2:NTF nuclei (in wild-type background) and beads before (D) and after (E) INTACT. Bar = 50 μm. Red, yellow and green arrowheads indicate nuclei-beads clumps, isolated beads, and isolated nuclei, respectively. Figure S3. Similarity in expression patterns between samples in the experiment. Read counts per gene were used to calculate the Poisson dissimilarity matrix between samples as implemented in the PoiClaClu package in R. Differences in color represent differences in expression profiles between samples and are represented in a heatmap. Figure S4. Somatic embryos. (A) Multiple somatic embryos emerging from embryogenic callus. Scale bar = 500 μm. (B) Optical longitudinal section of a somatic embryo (mPS-PI imaging technique). Scale bar = 50 μm. (DOCX 3601 kb

Mitochondrial Transcriptome Control and Intercompartment Cross-Talk During Plant Development

We address here organellar genetic regulation and intercompartment genome coordination. We developed earlier a strategy relying on a tRNA-like shuttle to mediate import of nuclear transgene-encoded custom RNAs into mitochondria in plants. In the present work, we used this strategy to drive trans-cleaving hammerhead ribozymes into the organelles, to knock down specific mitochondrial RNAs and analyze the regulatory impact. In a similar approach, the tRNA mimic was used to import into mitochondria in Arabidopsis thaliana the orf77, an RNA associated with cytoplasmic male sterility in maize and possessing sequence identities with the atp9 mitochondrial RNA. In both cases, inducible expression of the transgenes allowed to characterise early regulation and signaling responses triggered by these respective manipulations of the organellar transcriptome. The results imply that the mitochondrial transcriptome is tightly controlled by a “buffering” mechanism at the early and intermediate stages of plant development, a control that is released at later stages. On the other hand, high throughput analyses showed that knocking down a specific mitochondrial mRNA triggered a retrograde signaling and an anterograde nuclear transcriptome response involving a series of transcription factor genes and small RNAs. Our results strongly support transcriptome coordination mechanisms within the organelles and between the organelles and the nucleu

Hypomethylated poplars show higher tolerance to water deficit and highlight a dual role for DNA methylation in shoot meristem: regulation of stress response and genome integrity

As fixed and long living organisms subjected to repeated environmental stresses, trees have developed mechanisms such as phenotypic plasticity that help them to cope with fluctuating environmental conditions. Here, we tested the role DNA methylation as a hub of integration, linking plasticity and physiological response to water deficit in the shoot apical meristem of the model tree poplar ( Populus ). Using a reverse genetic approach, we compared hypomethylated RNAi- ddm1 lines to wild-type trees for drought tolerance. An integrative analysis was realized with phytohormone balance, methylomes, transcriptomes and mobilomes. Hypomethylated lines were more tolerant when subjected to moderate water deficit and were intrinsically more tolerant to drought-induced cavitation. The alteration of the DDM1 machinery induced variation in DNA methylation in a cytosine context dependent manner, both in genes and transposable elements. Hypomethylated lines subjected to water deficit showed altered expression of genes involved in phytohormone pathways, such as salicylic acid and modified hormonal balance. Several transposable elements showed stress- and/or line-specific patterns of reactivation, and we could detect copy number variations for two of them in stressed ddm1 lines. Overall, our data highlight two major roles for DNA methylation in the shoot apical meristem: control of stress response and plasticity through transduction of hormone signaling and maintenance of genome integrity through the control of transposable elements

RNAi suppression of DNA methylation affects the drought stress response and genome integrity in transgenic poplar

Trees are long-lived organisms that continuously adapt to their environments, a process in which epigenetic mechanisms are likely to play a key role. Via down regulation of the chromatin remodeler DECREASED IN DNA METHYLATION 1 (DDM1) in poplar (Populus tremula 9 Populus alba) RNAi lines, we examined how DNA methylation coordinates genomic and physiological responses to moderate water deficit.We compared the growth and drought response of two RNAi-ddm1 lines to wild-type (WT) trees under well-watered and water deficit/rewatering conditions, and analyzed their methylomes, transcriptomes, mobilomes and phytohormone contents in the shoot apical meristem. The RNAi-ddm1 lines were more tolerant to drought-induced cavitation but did not differ in height or stem diameter growth. About 5000 differentially methylated regions were consistently detected in both RNAi-ddm1 lines, colocalizing with 910 genes and 89 active transposable elements. Under water deficit conditions, 136 differentially expressed genes were found, including many involved in phytohormone pathways ; changes in phytohormone concentrations were also detected. Finally, the combination of hypomethylation and drought led to the mobility of two transposable elements. Our findings suggest major roles for DNA methylation in regulation of genes involved in hormone-related stress responses, and the maintenance of genome integrity through repression of transposable elements

RNAi suppression of DNA methylation affects the drought stress response and genome integrity in transgenic poplar

Trees are long-lived organisms that continuously adapt to their environments, a process in which epigenetic mechanisms are likely to play a key role. Via down regulation of the chromatin remodeler DECREASED IN DNA METHYLATION 1 (DDM1) in poplar (Populus tremula 9 Populus alba) RNAi lines, we examined how DNA methylation coordinates genomic and physiological responses to moderate water deficit.We compared the growth and drought response of two RNAi-ddm1 lines to wild-type (WT) trees under well-watered and water deficit/rewatering conditions, and analyzed their methylomes, transcriptomes, mobilomes and phytohormone contents in the shoot apical meristem. The RNAi-ddm1 lines were more tolerant to drought-induced cavitation but did not differ in height or stem diameter growth. About 5000 differentially methylated regions were consistently detected in both RNAi-ddm1 lines, colocalizing with 910 genes and 89 active transposable elements. Under water deficit conditions, 136 differentially expressed genes were found, including many involved in phytohormone pathways ; changes in phytohormone concentrations were also detected. Finally, the combination of hypomethylation and drought led to the mobility of two transposable elements. Our findings suggest major roles for DNA methylation in regulation of genes involved in hormone-related stress responses, and the maintenance of genome integrity through repression of transposable elements